EP2619227A1

EP2619227A1 - Super-humanized antibodies

Info

Publication number: EP2619227A1
Application number: EP10793275.8A
Authority: EP
Inventors: Philippe Thullier
Original assignee: ETAT FRANCAIS (MINISTERE de la DEFENSE) SERVICE DE SANTE DES ARMEES
Current assignee: ETAT FRANCAIS (MINISTERE de la DEFENSE) SERVICE DE SANTE DES ARMEES
Priority date: 2010-09-21
Filing date: 2010-09-21
Publication date: 2013-07-31
Also published as: CA2811462A1; US20130184440A1; WO2012038609A1

Abstract

The subject matter of the present invention is a method for preparing a germlined hypervariable region of an antibody directed against a target, and also the antibodies, antibody fragments, vectors and compositions comprising such a germlined hypervariable region.

Description

Hyperhumanized antibodies

The present invention relates to a process for the preparation of hyperhumanized antibodies.

Monoclonal antibodies represent a therapeutic class that is in constant development. The recent and considerable growth of recombinant monoclonal antibodies (Ac) is due in particular to the good tolerance of these molecules, which have evolved from murine Ac to human Ac, through intermediate generations consisting of chimerized and humanized Ac.

The logic of this evolution was to reduce the size of murine regions in the variable domains of these therapeutic Ac, especially in the framework regions (humanized Ac), to finally go to completely human antibodies. The improvement in tolerance that could then be observed (in particular a decrease in the frequency of appearance of therapeutic anti-Ab Ab and / or an increase in the lifetime of the therapeutic Aabs) reflects the accuracy of this approach, even if the tolerance Human immunoglobulin G (IgG) is not expected to be perfect, as evidenced by previous work on idiotypic networks, for example (Behn U. Idiotypic Networks: Toward a Renaissance? Immunol Rev 2007; 216: 142-52; Sfikakis PP The First Decade of Biology Tnf Antagonists in Clinical Practice: Learned Lessons, Unresolved Issues and Future Directions, Curr Dir Autoimmun 2010; 11: 180-210.)

The work of J. Foote's team (Hwang et al., Methods Vol.36, Issue 1, May 2005, pages 35-42) and P. Thullier (Pelat et al., J Mol Biol, 2008 Dec 31; 384 (5): 1400-7) have demonstrated the possibility of "germinalising"("germline-humanize") Ac in framework regions ("framework" or FR), respectively of murine origin or from non-human macaques. According to Pelât et al., The framework regions of non-human primate (PNH) acs have been "germinated", i.e., these regions have been mutated to make them very close to sequences encoded by human germ genes. . The advantage of this approach is that the sequences coded by the human germinal genes are part of the human "immunological self", and therefore are well tolerated during human therapeutic use. The antibody sequences encoded by human germ genes encode IgM but not IgG because IgG undergo affinity maturation, which is enabled by mutations to germinal genes but these mutations can be immunogenic. The mutated germ genes encoding IgG are called somatic genes.

Hypervariable regions (CDR), in turn, are highly exposed to the surface of the Ac, so as to interact directly with the antigens. They are therefore very likely to cause a humoral immunological response, even more than the framework regions. However, the interaction of the Ac with the antigen is highly dependent on the sequences of the hypervariable regions, and any mutation in these regions is highly likely to alter the affinity of the Ac for its antigen.

The use of Ac in a given treatment always raises the question of

Immunogenicity.

There is therefore a need for Ac having a good tolerance in clinical use therefore low immunogenicity in humans, without significantly altering their affinity for the antigen. To reduce or even cancel the immunogenicity of the Ac during their therapeutic use, while maintaining their affinity, is the object of the invention.

The present invention relates to a strategy for decreasing immunogenicity of antibodies by mutating the somatic genes encoding hypervariable regions of recombinant antibodies to approximate human germ genes encoding these regions.

The inventors have recently discovered that, surprisingly, the mutation of the CDR regions so as to make them very close to the sequences coded by the human germinal genes makes it possible to meet these expectations. Indeed, when the hypervariable regions of Ac are mutated to be made as close as possible to the sequences coded by the human germinal genes, the problem of immunogenicity is avoided or limited since the sequences thus mutated are closer to the "self". and, surprisingly, such mutations are widely possible while retaining an affinity of the Ac for its antigen comparable to the affinity of the initial Ac. Improvement of tolerance in humans correlates with decreased immune responses to antibodies and modified, because of its greater proximity to the human "immunological self" compared to the parental antibody.

The subject of the present invention is therefore a process for preparing a germinated hypervariable region of antibodies directed against a target, comprising the following steps:

a) obtaining the peptide sequence of a hypervariable region of an antibody or a fragment of Mammalian antibody, with the exception of a human IgM, said mammal having a human homologous sequence, said antibody or fragment of a antibody being directed against said target;

b) a comparison between the peptide sequence of the hypervariable region obtained in a) and the peptide sequences coded by the human V, (D), J genes, in order to identify at least one germinal gene V, (D) or J human coding the peptide sequence closest to the peptide sequence of the hypervariable region obtained in a);

c) for each amino acid different between the peptide sequence obtained in a) and the closest peptide sequence obtained in b), substitution by in vitro mutagenesis of each amino acid of the peptide sequence of the hypervariable region obtained in a), by the corresponding amino acid of the peptide sequence encoded by the germinal gene V, (D) or human J identified in b), to obtain a series of mutated hypervariable regions of Mammal, each peptide sequence of hypervariable region comprising at least a mutation ;

d) selecting mutated hypervariable regions obtained in c) having an affinity for said target comparable to or greater than that of the hypervariable region of antibody or antibody fragment obtained in a); and

e) optionally, preparing the peptide sequence of the hypervariable region of antibody or of an antibody fragment directed against said target comprising all or part of the mutations present in at least one peptide sequence of the selection obtained in d).

Steps a) and b) may also consist of looking for the nucleotide sequences closest to the nucleotide sequences encoding the sequences peptides of the hypervariable regions of the Mammalian antibody. Also, the present invention also relates to a method for preparing a hypervariable hypervariable region of antibody directed against a target (hereinafter "alternative method"), comprising the following steps:

a) obtaining the nucleotide sequence of a hypervariable region of an antibody or a fragment of Mammalian antibody, with the exception of a human IgM, said mammal having a human homologous sequence, said antibody or fragment thereof antibody being directed against said target;

b) a comparison between the nucleotide sequence of the hypervariable region obtained in a) and the nucleotide sequences coded by the human V, (D), J genes, in order to identify at least one germinal gene V, (D) or J human coding the nucleotide sequence closest to the nucleotide sequence of the hypervariable region obtained in a);

c) for each nucleotide different between the nucleotide sequence obtained in a) and the closest nucleotide sequence obtained in b), substitution by in vitro directed mutagenesis of each nucleotide of the nucleotide sequence of the hypervariable region obtained in a), by the corresponding nucleotide of the nucleotide sequence encoded by the germinal gene V, (D) or human J identified in b), in order to obtain a series of nucleotide sequences of mutated hypervariable regions of Mammal, each nucleotide sequence of hypervariable region comprising at least one mutation;

d) translating nucleotide sequences of mutated hypervariable regions obtained in c) and selecting mutated hypervariable regions having an affinity for said target comparable to or greater than that of the hypervariable region of antibody or antibody fragment obtained in a); and

e) optionally, preparing the peptide sequence of the hypervariable region of antibody or of an antibody fragment directed against said target comprising all or part of the mutations present in at least one peptide sequence of the selection obtained in d). The process according to the invention is an in vitro process; he uses the techniques of molecular biology. This process corresponds to the germinalization of the hypervariable regions of antibodies. To minimize immunogenicity, step e) preferably comprises preparing the peptide sequence of the hypervariable region of antibody or antibody fragment directed against said target, identical to the nearest sequence encoded by at least one human germinal gene V, D or J identified in step b). Also, in order to minimize the immunogenicity of a variable antibody region, germinalization is conducted simultaneously in the framework regions and hypervariable regions, without explicitly distinguishing either type of region. The present invention also relates to a hypervariable region of germination of antibody obtainable by said method. The subject of the invention is also an antibody or antibody fragment comprising the hypervariable germinalised region thus obtained.

Another subject of the invention is the nucleic sequence encoding said germinated hypervariable region or said antibody or said antibody fragment. This nucleic sequence can be included in a vector.

Finally, the subject of the present invention is a composition comprising said germinated hypervariable region or said antibody or said antibody fragment.

The present invention also relates to the use of said germinated hypervariable region or said antibody or said antibody fragment in therapy.

The present invention will be better understood with the aid of the following definitions.

As is well known, only a portion of the antibody, the variable region, is involved in the binding of the antibody to its epitope. The constant regions of the antibody activate the immune effectors, phagocytes or killer cells, and the complement; these constant regions are not involved in antigen binding. The antibodies comprise two identical heavy chains associated with two identical light chains, kappa or lambda. An antibody whose constant region (Fc) has been enzymatically cleaved so as to preserve the hinge region is designated as an F (ab ') 2 fragment and retains both antigen binding sites.

Likewise, an antibody whose constant region, including the hinge region has been enzymatically cleaved, or which has been produced without this region, is designated as a Fab fragment and retains one of the two antigen binding sites. The Fab fragments consist of a light chain that is covalently linked to a portion of the heavy chain called Fd.

In the variable region are the complementarity determining regions (CDRs), or hypervariable regions, which interact directly with the antigen. There is also a second type of region, called framework regions (FRs), which maintain the tertiary structure of CDRs. These framework regions are fairly specific to the species where the antibody was produced. In the Fd fragment of the heavy chain and in the light chain, there are four framework regions (FR1 to 4) separated respectively by three CDRs (CDR1 to 3) on each chain.

The variable region of an antibody is encoded by several genes: variable segment (V), diversity (D) and junction (J) genes. The variable region of an antibody heavy chain is in particular encoded by a V gene, a D gene and a J gene; the variable region of a light chain is in particular coded by a V gene and a J gene, but does not include a D gene. By recombination of the human germinal genes V, D and J, a VDJ or VJ sequence is obtained, to which Residues can be added to the V / D and D / J (heavy chain) junctions or to the V / J (light chain) junction, respectively coding for the variable parts of the heavy and light chains.

In humans, heavy chain genes (IGH locus), kappa light chain genes (IGK locus), and lambda light chain genes (IGL locus) are located on chromosomes 14, 2, and 22, respectively. recombination of IGH, IGK and IGL locus genes during B cell differentiation, antibodies are synthesized.

In humans, the IGK locus comprises 76 IGKV (variable) genes, of which 34 to 37 are functional and belong to 5 subgroups; the IGL locus contains 70 to 74 genes IGLV (variables) of which 29 to 35 are functional and belong to 10 subgroups. There are five IGKJ genes (junction) located in 3 'IGKV genes. Finally, the IGH locus comprises 123 to 129 IGHV genes according to the haplotypes, of which 38 to 46 are functional and belong to 6 or 7 subgroups. Twenty-seven D (diversity) genes, including 23 functional and nine JH genes, including six functional genes, have been described (Molecular Genetics of Immunoglobulins, Prof.Marie-Paule LEFRANC and Gérard LEFRANC, IMGT Education).

The term "antibody" refers to an immunoglobulin molecule having the ability to bind specifically to a particular antigen. Examples of well-known antibody or immunoglobulin fragments are, for example, F (ab ') 2, F (ab) 2, Fab, Fab', Fv, scFv, diabody, dAb and Fd fragments, or a heavy chain or a light chain, a VH or a VL. The term "hypervariable region" refers to a CDR: 3 CDRs are present on the variable part of the heavy chain, and 3 CDRs are present on the variable part of the light chain. These hypervariable regions are in close contact with the antigen. By CDR, one thus understands as well one of the peptide sequences present on the heavy chain (ie CDR1, CDR2 or CDR3 of VH), that one of the peptide sequences present on the light chain (ie CDR1, CDR2 or CDR3 of VL). In the present application, when we speak of "peptide sequence of a hypervariable region", we mean the peptide sequence of a CDR of the heavy chain or of the light chain. The 3 CDRs present on the variable part of the heavy chain and the 3 CDRs present on the variable part of the light chain form the site of attachment to the antigen. If several delimitations of the CDRs can be proposed as part of their scientific definition, the delimitation encompassing the maximum size of each of the CDRs must preferably be selected according to the present invention. Any other definition is nevertheless acceptable. The mammal from which the peptide (or nucleotide) sequence of the hypervariable region of antibody or antibody fragment used in a) originates must have a homologous human sequence. Indeed, to obtain a hypervariable region When germinalis is administered to humans, it is important that the initial sequence has a human homologous sequence, which serves as a reference for carrying out the method according to the invention. By antibody homologous to a human antibody is meant an antibody derived from a common ancestral sequence (common ancestor) with the human antibody. The Mammal CDR is preferably a Primate CDR. Primates can be human or non-human, including Cercopithecidae (Cercopithecidae) and Hominids (Hominidae). Preferably, mammals are selected from macaque, man, chimpanzee, bonobo, gorilla, orangutan, and baboon. Preferably, mammals are selected from macaque and man.

The term "human germinal gene" refers to any human gene present in germ cells (spermatozoa, ova). Such a gene comprises an unmodified nucleotide sequence, i.e. having not undergone any mutation related to affinity maturation. The germinal genes V, D and J, especially human, within the meaning of the invention, have undergone no recombination and no mutation related affinity maturation: they are in the germinal configuration.

Affinity maturation is based on two processes:

somatic mutations that affect all the gene segments that encode the variable regions of the antibodies, i.e. the variable segments (V), the diversity segments (D) and the joining segments (J);

and antigen-directed selection which leads to the expansion of clones which, following mutations, express the highest affinity antibodies. Also, a human germinal V, D or J gene has not undergone any somatic mutation related to affinity maturation, and corresponds to the initial sequence present in human germ cells. Since the invention relates to antibodies, such human germinal genes are understood in the present invention as those encoding human IgM. The human IgM are in fact coded in their variable part by recombinant germinal gene sequences VDJ (heavy chains) and VJ (light chains).

The invention is thus applicable to any antibody that is not encoded by human germ genes, therefore to any antibody except human IgM. By "human germinal gene coding for the nearest peptide sequence" of a given sequence is meant the human germinal gene coding for the peptide sequence (or nucleotide for the alternative method) which has the greatest "percentage of identity" with the given sequence, or the largest "percentage of similarity" with the given sequence. By abbreviation, in the remainder of the present application, reference will be made indifferently to the term "human germinal gene coding for the nearest peptide sequence (or nucleotide for the alternative method)" or "nearest human germinal gene".

The percentage of similarity of peptide sequences takes into account the identities between amino acids, as well as the similar physicochemical properties between certain amino acids. It is said that amino acids have similar physicochemical properties if the substitution of one amino acid for another does not interfere with the function of the protein; this is called conservative substitution. For example, the hydrophobic amino acid valine can be exchanged for leucine. The percentage of similarity is calculated as the percentage of identity, detailed below, but takes into account the identity between amino acids and conservative substitutions.

The percentage of peptide sequence identity is determined by comparing 2 optimally aligned sequences on a comparison window, in which the amino acid sequence portion may comprise additions or deletions (ie gaps) compared to the reference sequence (which does not include addition or deletion) for optimal alignment of the 2 sequences. The percent identity can be calculated by determining the number of positions at which identical amino acid residues appear in both sequences, to obtain the number of identical positions, dividing the number of identical positions by the total number of positions in the two sequences. the comparison window, and multiplying by 100 to get the percentage of sequence identity.

Alternatively, the percentage of identity can be calculated by determining the number of positions at which either the identical amino acid residue between the two sequences, or the amino acid residue is aligned with a gap to obtain the number of identical positions, dividing the number of identical positions by the total number of positions in the comparison window, and multiplying by 100 to obtain the percentage of sequence identity. Many algorithms are available to align 2 sequences. The optimal alignment of sequences can be carried out in particular by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. ScL USA 85: 2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see Current Protocols in Molecular Biology, FM Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms include BLAST and BLAST 2.0, described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. A program for conducting BLAST analyzes is available on the National Center for Biotechnology Information website. For example, a sequence alignment and percent sequence identity determination can use the BESTFIT or GAP program in the GCG Wisconsin Software package (Accelrys, Madison WI), using the default settings.

The percent identity of nucleotide sequences is determined as described for the peptide sequences, but this time using nucleotide sequences.

The term "germinalised hypervariable region" refers to a Mammalian CDR, preferably Primate, wherein certain amino acids (or nucleotides for the alternative method) have been substituted by the amino acids (or nucleotides for the alternative method) shown in identical position (s) in the peptide sequence (or nucleotide for the alternative method) encoded by the nearest human germinal gene.

The K _D affinity of an antibody can be measured by conventional techniques known to those skilled in the art. Comparable affinity between 2 antibodies for a given target is when the affinity change does not cause a functional change in the antibody for therapeutic use (eg, no difference in toxin neutralization or cytotoxicity to target cells, between the initial antibody, ie at the base of step a), and the antibody, in particular IgG, obtained by the process according to the invention and proposed in therapy). Preferably, we speak of comparable affinities between 2 antibodies for a given target when their K _Ds vary by at most a factor of 100, preferably 50, preferably 10, preferably 5, preferably 2. Indeed, in some In this case, the activity of the antibody depends little on the affinity of its antigen binding sites.

The affinity of an antibody A is greater than the affinity of an antibody B for the same target if the K _D of A is at least 150 times smaller, preferably at least 500 times smaller, preferably at least 500 times smaller. 1000 times smaller than the K _D of antibody B.

The affinity constant K _D can be calculated from the association and dissociation constants measured in real time by surface plasmon resonance as explained by the protocols of the Biacore devices (General Electric Healthcare) or directly by competition ELISA. , for example.

The method according to the present invention thus provides germinalised hypervariable regions. Preferably, said hypervariable region (s) germinated is (are) present in antibody fragments of F (ab ') 2, Fab, Fv, scFv, diabody type, dAb and Fd, or in chimeric or fully human antibodies in which the Fc portion of the antibody is derived from human or non-human homologous sequences. These antibodies or antibody fragments may comprise a single germinated CDR, 2 germinated CDRs or the 3 CDRs of each of the heavy and / or light chains may be germinated (in total from 1 to 6 CDRs per antigen binding site , and from 1 to 12 CDRs per IgG, for example).

According to a mode of the invention, the Fc part of the antibody can be chosen so as to produce IgA, IgM or IgG.

According to a preferred embodiment of the invention, when the germinalised hypervariable region is present in an antibody, this antibody has a Fc portion of human origin. Such whole antibodies are preferred for administration in humans because they have a longer half-life than antibody fragments such as Fabs, and are more suitable for intravenous, intraperitoneal, intramuscular, subcutaneous administration. -cutaneous or transdermal. In some embodiments of the invention, antibody fragments such as Fab fragments are preferred for the following reasons:

i) since the Fab fragments have only one antigen binding site, large immune complexes can not form,

ii) the absence of Fc region prevents the appearance of an inflammatory reaction activated by Fc, such as the activation of the complement cascade,

(iii) the tissue penetration of a small Fab molecule is easier, and

iv) The production of Fabs is easily performed at low cost in bacteria such as E. coli.

Thus, in one aspect, the method according to the present invention provides Fabs having at least one germinated hypervariable region, or fragments thereof that are smaller or larger than Fabs or epitope binding peptides.

The method according to the present invention makes it possible to obtain antibodies and antibody fragments having a comparable or improved affinity compared to the initial mammalian antibody, preferably Primate, as well as better tolerance by the human immune system. Such a "germinated" antibody has the advantage of not or less inducing an immune response against itself, and to have a longer half-life and to induce fewer harmful side effects for the subject being treated.

The method according to the invention comprises a step a) of obtaining the peptide sequence (or nucleotide for the alternative method) of a hypervariable region of an antibody or Mammalian antibody fragment, preferably of Primate, said antibody or antibody fragment being distinct from a human IgM, said antibody or antibody fragment being directed against a target. As indicated above, the mammal chosen as the starting model has a human homologous sequence; this means that to obtain a hypervariable germline region of antibody directed against a target, the mammal chosen as the starting model must have an antibody directed against said target which is a homologue of a human antibody. This step a) can be carried out according to any known method of the prior art, in particular by sequencing from the native antibody or its fragment, or by sequencing the DNA encoding this native antibody or its fragment, and then translating the nucleotide sequence.

The peptide (or nucleotide) sequence of the hypervariable region is specific to a given target.

The sequence obtained in step a) is preferably a sequence of a hypervariable region of antibody or human antibody fragment or macaque, with the exception of a human IgM. Then, the peptide sequence of the hypervariable region obtained in a) is compared with the peptide sequences encoded by the germinal genes V, D and J humans. The peptide sequences encoded by the human V, D, and J germ-lines correspond to the peptide sequences of the heavy and light chain variable regions of antibodies that have not undergone affinity maturation. This comparison aims to identify at least one germinal gene V, D or J human nearest. The purpose of step b) is to identify, among the multitude of existing germ genes V, D and J, at least one germinal gene V, D or J having the highest percentage of identity with the sequence obtained. in a). Indeed, the peptide sequence encoded by the germinal gene V, D or J which has the highest percentage of identity with the hypervariable region sequence obtained in a) corresponds to the peptide sequence closest to the starting sequence. It therefore determines the closest human germinal gene, which is obtained at the end of step b).

Alternatively, when step a) uses nucleotide sequences (alternative method), it is also possible to compare, during this step b), the nucleotide sequences between them, and to mutate the hypervariable regions of the antibody to be germinated ( step c)) to increase proximity to the V (D) J genes

Preferably, step b) comprises the comparison between the peptide sequence of the hypervariable region obtained in a) and the peptide sequences encoded by the human germinal genes V, D and J.

Preferably, step b) comprises the comparison between the sequence obtained in a) and the peptide sequences encoded by all the human V, D and J germinal genes, in order to identify the V gene, the J gene and possibly the gene. D (ie in the case of a heavy chain) encoding the peptide sequences closest to the sequence obtained in a). With this step b), the human germinal genes V, J and possibly D with the highest identity percentages (therefore the closest) with the sequence obtained in a) are identified.

Step b) can be performed using online comparison tools, such as VQuest or DomainGapAlign of the IMGT server (htp: // im gt.cines.fr); it can also be done by other means, including manually. These tools indicate the peptide sequences encoded by the germinal genes V, D and J that are closest to each sequence obtained in a). Such an analysis also makes it possible to locate the differences between the peptide sequences obtained in a) and the peptide sequences coded by the human germinal genes.

Steps a) and b) can be performed on the heavy chain and / or the light chain of the hypervariable region.

The hypervariable region peptide sequence of antibody or antibody fragment obtained in a) is then substituted, for each position on which the amino acid differs between the sequence obtained in a) and the peptide sequence encoded by the germinal gene V , D or J nearest human (obtained in b)), by in vitro directed mutagenesis, by the corresponding amino acid of the latter sequence (step c)). For example, if a macaque antibody hypervariable region sequence is prepared in a), step c) may consist of a substitution, for each relevant amino acid, of the original amino acid of the macaque sequence by the corresponding amino acid of the peptide sequence encoded by the nearest germinal gene V, D or J: there is therefore a change in the sequence of the macaque hypervariable region to the human germinal sequence.

This step of site-directed mutagenesis can be done by any appropriate method, such as spot mutation (s) directed (s) or gene synthesis for example.

This step c) can be carried out by mutating the relevant amino acids one by one, or by mutating them in groups (s). At the end of step c), a series of hypervariable regions of antibodies or antibody fragments are obtained, each of these hypervariable regions comprising one or more mutations. In the example of the preceding paragraph, at the end of step c), it is thus possible to obtain a series of hypervariable regions of macaque origin, each comprising a mutation corresponding to the amino acid of the peptide sequence coded by the gene. germinal V, (D) or nearest human J.

Alternatively, when step a) uses nucleotide sequences (alternative method), at the end of step c) a series of nucleotide sequences of hypervariable regions of antibodies or antibody fragments are obtained, each of these hypervariable regions comprising one or more mutations.

From the series obtained in step c), it is then necessary to select the hypervariable regions of antibodies or antibody fragments having a target affinity comparable to or greater than the affinity of the initial hypervariable region (obtained in a). ) for the same target: this is step d). Indeed, step d) aims at selecting only mutated hypervariable regions (ie each comprising at least one point mutation that brings it closer to the nearest human germinal gene) having a comparable or greater affinity for the target, compared to the initial hypervariable region. This affinity can be measured by Biacore device or any equivalent technique as indicated above.

At the end of step d), a series of mutated hypervariable regions are obtained which have substantially the same affinity, or a higher affinity for the target than the initial hypervariable region.

Alternatively, when step a) uses nucleotide sequences (alternative method), step d) comprises the translation of the nucleotide sequences of mutated hypervariable regions obtained in c), then the selection of hypervariable regions of antibodies or fragments of nucleotides. antibody having a target affinity comparable to or greater than the affinity of the initial hypervariable region (obtained in a) for the same target.

From this selection, one can optionally add a step e): this step e) comprises the preparation of a hypervariable region having all or part of the mutations present in at least one peptide sequence selected in step d). Preferably, this step e) comprises the preparation of a hypervariable region having all the mutations present in all the peptide sequences of the selection of step d).

The product obtained at the end of step e) thus corresponds to a synthetic hypervariable region sequence of Mammalian antibody comprising one or more point mutations that bring said sequence of the nearest human germinal gene closer together. Also, preferably, in fine, one obtains a hypervariable region having a strong identity or a strong similarity with the closest human germinal genes, and which has the affinity of the initial antibody (ie at the base of the step at)).

Preferably, the method according to the invention also comprises, after step d) or e) (when the latter is performed), a step f) of evaluating the germinated hypervariable region obtained. This step f) may comprise the calculation of the percentage of similarity with the sequence encoded by the nearest human germinal gene (or "germinality index") for the initial peptide sequence obtained in a), and for the final peptide sequence obtained in d) or e). The germinality index according to the invention can be measured by analogy as indicated in Pelat et al, Germline Humanization of a Non-human Primate Antibody that Neutralizes the Anthrax Toxin, by Vitro and in Silico Engineering, J. Mol. Biol. (2008) 384, 1400-1407. Pelat et al describes the notion of germinality index for FR framework regions only; it is possible to extrapolate this calculation in a similar way for the hypervariable regions, and also for the variable regions. Briefly, the germinality index according to the present invention is calculated as follows: the nearest human germinal gene is translated, and the percentage of identical amino acids between the translated sequence and the peptide sequence (s) (s) ) of hypervariable region obtained in a) (or d) or e)) is. calculated and called germinality index. Step f) can also be performed by calculating H-scores and G-scores. These scores are derived from Z-score, which indicates the degree of "humanness" of a sequence: a Z-score of 0 corresponds to a sequence that is on average similar to the human sequence repertoire, a Z-positive score corresponds to sequences that are on average strongly identical to human sequences, and a negative Z-score corresponds to sequences that are on average less typical of human sequences (Abhinandan et al, Analyzing the "Degree of Humanness" of Antibody Sequences, J. Mol Biol (2007) 369, 852-862).

The H-score (for "humanness score") corresponds to the Z-score calculated with respect to all the human repertoire expressed and represented in the Kabat sequence library, and the G-score (for "germline-derived score") corresponds to Z-score calculated with respect to each part of this directory which belongs to the same family, then averaged for all these parts. H-score and G-score can be measured as indicated in Thullier et al., The Humanness of Macaque Antibody Sequences, J. Mol. Biol. (2010).

When the method according to the invention comprises a step f), this step can comprise the calculation:

the germinality index of the peptide sequence obtained in a) and the germinality index of the peptide sequence obtained in d) or e); and or

the H-score or G-score of the peptide sequence obtained in a) and the peptide sequence obtained in d) or e),

then the respective comparison between these values.

The germinality index should normally be increased between the initial peptide sequence obtained in a) and the final peptide sequence obtained in d) or e) by the method according to the invention. Similarly, the H- and G-scores should normally be increased between the initial peptide sequence obtained in a) and the final peptide sequence obtained in d) or e) by the method according to the invention.

The subject of the present invention is also a germinated hypervariable region of antibody obtainable by the method described above.

The subject of the invention is also an antibody or antibody fragment comprising the hypervariable germinalised region thus obtained. Such an antibody or antibody fragment may comprise a single germinated CDR, but may also include 2 or 3 CDRs of each of the heavy and / or light chains. Such an antibody or antibody fragment may also comprise, in addition to one or more germinated CDRs, one or more framework regions (FR) also germinalised. Another subject of the invention is a vector comprising the nucleic sequence encoding said germinated hypervariable region or said antibody or said antibody fragment.

These nucleic acids may be included in a recombinant vector for cloning or for the expression of the antibodies of the invention.

The present invention includes all recombinant vectors containing coding sequences for transformation, transfection or eukaryotic or prokaryotic gene therapy. Such vectors may be prepared according to conventional molecular biology techniques and will further include a suitable promoter, optionally a signal sequence for export or secretion, and regulatory sequences necessary for transcription of the nucleotide sequence.

A fusion polypeptide may be useful for the purification of the antibodies of the present invention. The fusion domain may for example include a poly-histidine tail which allows purification on Ni + columns, or a filamentous phage membrane anchor which is particularly useful for bank screening, according to the "phage display" technology.

One of the appropriate vectors in the context of the invention is a recombinant DNA molecule adapted to receive and express a first and a second DNA sequence, so as to allow the expression of heterodimeric antibodies such as an antibody of complete length or fragments F (ab ') 2 or Fab according to the invention. Such a vector provides a system for independently cloning the two DNA sequences into two separate cassettes present in the vector, so as to form two separate cistrons for the expression of a first and a second polypeptide of the heterodimeric antibody. . Such an expression vector is called a di-cistronic vector.

The modified antibodies of the present invention can be produced in eukaryotic cells such as CHO or human or murine hybridomas for example, as well as in plant cells. The subject of the present invention is also host cells, prokaryotic or eukaryotic, comprising a vector according to the invention.

Finally, the subject of the present invention is a composition, in particular a pharmaceutical composition, comprising said germinated hypervariable region or said antibody or said antibody fragment.

Said pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier. Such a vehicle corresponds to the meaning of the invention to a non-toxic material which does not interfere with the effectiveness of the biological activity of the active ingredients of the composition. The term "pharmaceutically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, a cell culture, a tissue or an organism. The characteristics of the vehicle will depend on the mode of administration. The antibody or germinated antibody fragment according to the invention can be labeled. Examples of markers include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Methods of binding a marker to an antibody are well known to those skilled in the art.

Another labeling technique involves coupling the antibody to low molecular weight haptens, which haptens can be specifically modified by means of a second reaction. Examples of haptens are biotin, which reacts with avidin, or dinitrophenol, pyridoxal or fluorescein, which can react with specific anti-hapten antibodies.

The present invention relates to a method of treating a subject, preferably a human, in which a therapeutically effective amount of an antibody or an antibody fragment according to the invention is administered to said subject.

A therapeutically effective amount is an amount sufficient to decrease the symptoms of the disease and the course of the infection. This amount may vary with the age, sex of the subject and the stage of the disease and will be determined by those skilled in the art. A therapeutically effective amount may vary between 0.01 mg / kg and 50 mg / kg, preferably between 0.1 mg / kg and 20 mg / kg, and more preferably between 0.1 mg / kg and 2 mg / kg, in one or more administrations daily, for one or more days.

The mode of administration may be by injection or by gradual infusion. The injection can be intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal.

Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, or injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcohol / water solutions, emulsions or suspensions.

The present invention will be better understood with the aid of the additional description which follows, which refers to an example of obtaining antibodies according to the invention.

In the examples which follow, given by way of illustration, reference will be made to the appended figures:

Figure 1: Pearl necklace pattern of the variable region of the heavy chain and the variable region of the light chain of the 45RCA antibody.

The IMGT representation in pearl necklace is made in accordance with the IMGT numbering.

Materials and methods

E. coli strains

The following E. coli strains were used:

XL1 (Stratagene, La jolla, CA): recAl, endAl, gyrA96 thi-1 hsdR17 sup E44 relAl lac [F'proAB lacIqZAM15 Tn10 (Tetr)] - SURE (Stratagene): el4 (MCRA) A (mcrCB-hsdSMR-mrr) 171 Endal supE44 thi-1 gyrA96 RELAL Lake recB recJ SBCC UMUC :: Tn5 (Kan r) uvrC [F ^'pro AB lacIqZAM15 Tn (Tet r)]

- HB2151, used for the expression of soluble scFv.

Antibodies and toxins

The antibody used is a very high affinity (41 pM), ricin-neutralizing PNH scFv called 43RCA (T. Pelat et al, BMC Biotechnology 2009, 9: 60).

The ricin A chain, injected into the macaque, was purchased from Vector Labs, as was the complete ricin.

Phage antibody selection

The phage-scFv particles were purified and concentrated from 50 ml of culture by PEG precipitation, and then resuspended in 3 ml of PBS-BSA 1% -azide 0.02% and filtered through a 0 filter. 45 μιη. The titer of this phage preparation was about ¹⁰ pfu / ml. The scFv-phages were subjected to three cycles of infection-selection-recovery as previously described (Andris-Widhopf, Rader et al., 2000). Expression of soluble scFv, periplasmic extraction and purification

Each DNA variant was transformed into bacteria of the E strain. coli called HB2151, made chemically competent. The cells were cultured at 30 ° C., shaken at 250 rpm in IL of SB medium containing 50 μg / ml of carbenicillin and 0.1% of glucose. When the culture reached an A ₆ of 1.5, induction with 1 mM IPTG was performed for 18h at 22 ° C.

The scFvs were extracted with polymixin B sulfate (Sigma) and purified on a nickel column (Ni-NTA spin column, QIAGEN, Valencia, CA) according to the manufacturer's instructions, and then dialyzed with IX PBS at 4 ° C for 3h. Quantization of scFv

The purity of the Fab was tested by SDS-PAGE and its concentration was determined using the Quantity One® software (Biorad). Real time measurement of surface plasmon resonance (SPR)

The kinetics of the interaction between ricin and scFv obtained previously were determined using the Biacore X SPR system (General Electric Healthcare). Ricin was immobilized on a CM5 sensitive chip using an amide coupling procedure by injection of 30 μΐ of 2 μg / ml of ricin in 10 mM sodium acetate pH 4.5. To minimize the likelihood of relinking, K _D was measured using a high flow rate (30 μΐ / min) and a minimal amount of coupled antigen (approximately 500 RU, resonance units). The binding rate of different concentrations of scFv ranging from 5 to 400 nM in PBS was determined at a flow rate of 30 μΐ / min. The link data was introduced into a 1: 1 langmuir model of the BIA evaluation software. The association and dissociation constants (k _on and k _Qff respectively) for the binding of scFv to ricin were determined at 35 ° C.

Sequence analysis

Sequences of the heavy and light chain variable regions of the selected clones were determined by Genome Express (Meylan, France) using primers Mkmyc and MkpelB, (Kirsch et al., 2005). The sequences were analyzed online, using the IMGT system (http: /imgt.cines.fr).

Results

Obtaining germinalised antibodies in their FR framework regions

ScFv 43RCA was analyzed using the DomainGapAlign tool, which indicated the human germ genes encoding sequences closest to the 43RCA peptide sequences. These genes were IGKV1-5 * 01 and IGKJ4 * 01 for the light chain, and IGHV3-11 * 01 and IGHJ5 * 01 for the heavy chain (DomainGapAlign does not search for D genes).

The germinalization of the framework regions (or "super-humanization") took place as shown in Table 1. Table 1: Mutations corresponding to the super-humanization of 43RCA

NB: the amino acids are designated by the one-letter code It has thus been observed that four mutations (in position 3,4,13,14) alter the affinity; they are shown in italics and in bold in Table 1. The other fifteen mutations were associated in a synthetic gene coding for the super-human variant, whose affinity for ricin was measured at 2.7 × 10 ^-11 M (27 pM).

Starting from this super-humanized variant, the germinalization of the hypervariable CDR regions was performed.

Obtaining germinalised antibodies in their FR framework regions and in their CDR regions

Starting from the super-humanized variant of 43RCA, the mutations corresponding to the germinalization in the hypervariable regions, and appearing in Table 2, were carried out ("hyper-humanization"). Table 2: Mutations corresponding to the hyper-humanization of 43RCA

It has been observed that five mutations (positions 2, 4, 12, 13, 14) alter the affinity; they are in italics and bold. The ten other mutations were combined in a synthetic gene encoding the variant super- and hyper-humanized, whose affinity for ricin was measured to 8,9 10 ^"11 M (89 pM). Eleven fifteen on positions therefore could be mutated without significant alteration of the reactivity of the Ac, compared to the affinity of parental scFv.

The results of this work can be evaluated by the Z-score measurement (KR Abhinandan & A. Martin, 2007), or the germinality index (T. Pelât & al, 2008) extended to hypervariable regions, or parameters derived from the two previous ones.

Claims

A process for preparing a hypervariable region of germinalised antibodies directed against a target, comprising the steps of:

a) obtaining the peptide sequence of a hypervariable region of an antibody or a fragment of Mammalian antibody, with the exception of a human IgM, said mammal having a human homologous sequence, and said antibody or fragment antibody being directed against said target;

2. Method according to claim 1, characterized in that the hypervariable region of Mammal comes from Primate, preferably Cercopithecidae (Cercopithecidae) or Hominids (Hominidae), preferably macaque or human.

3. Method according to claim 1 or 2, characterized in that it comprises a step f) of calculation:

then the respective comparison between these values.

4. Method according to one of claims 1 to 3, characterized in that the antibody fragment is selected from a heavy chain, a light chain, a VL, a VH, a

Fab, Fab ', F (ab) 2, F (ab') 2, scFv, diabody, and dAb.

5. Method according to one of claims 1 to 4, characterized in that step b) comprises the comparison between the peptide sequence of the hypervariable region obtained in a), and the peptide sequences encoded by all the germinal genes V, D, J humans, to identify human germ genes V, J and possibly D coding the peptide sequences closest to the peptide sequence of the hypervariable region obtained in a).

6. Method according to one of claims 1 to 5, characterized in that step e) comprises the preparation of the peptide sequence of the hypervariable region of antibody or antibody fragment directed against said target comprising all the mutations. present in all the peptide sequences of the selection obtained in d).

7. Hypervariable germinated antibody region obtainable by the method according to one of claims 1 to 6.

An antibody or antibody fragment comprising the germinalised hypervariable region of claim 7.

A nucleic acid sequence encoding the hypervariable germinated region of claim 7 or the antibody or antibody fragment of claim 8.

A vector comprising a nucleic acid sequence according to claim 9.

A composition comprising the hypervariable germinated region of claim 7 or the antibody or antibody fragment of claim 8 or the vector of claim 10.

The hypervariable germinated region of claim 7 or the antibody or antibody fragment of claim 8 for use in therapy.

A process for preparing a hypervariable hypervariable region of antibody directed against a target, comprising the steps of: